Quantitative and equipment-free paper-based agglutination assay of bacterial cells

Abstract

Background: point-of-care (POC) tests are useful for bedside/home applications, emergencies, frequent follow-ups, and resource-limited areas. Limited quantitative and equipment-free POC assays have been reported. This study aims to develop, validate, and apply a simple, quantitative, paper-based POC assay. Methods: wax-channeled paper treated with specific anti-Brucella and anti-Salmonella antibodies was used for distance-based chromatographic elution of stained bacterial cell agglutinations. Results: a qualitative paper-based agglutination POC test was developed using color intensity, tail appearance, and “+/−” signs that clearly distinguish the positive and negative results. The optimization of the test for paper type, microfluidic channel design, antibody and bacterial cell concentrations, and elution methods was carried out. Quantitative assay transformation was successfully developed using the color intensity of the original reaction zone, intensity of elution tail, and distance-based migration that correspond to bacterial agglutination size. The migration distance of eluted bacterial agglutination bands corresponds to the target concentration with good linearity and minimal variability. Reporting of colored band migration with numbers using microfluidic patterns was used to enhance non-technical end-user applications. A distance-based POC assay prototype was then successfully used for the accurate detection of known and unknown samples in comparison with standard assays. Conclusions: the migration distance of an eluted stained bacterial agglutination correlated with anti-bacterial antibody concentrations. A simple, cheap, quantitative, and equipment-free paper-based POC assay of bacterial cell agglutination was developed. This test can be used for simple “+/−” results, thermometer-like quantification, or text reporting with numbers corresponding to target concentrations. The assay has extended applications to different human disease biomarkers.